1. The use of bacterial host strains in biotechnology
Medical biotechnology now encompasses a broad range of medical technologies that have veterinary and human applications. At the crux of this technology is the use of recombinant DNA, molecular biochemistry and immunochemical techniques, which allow the identification, characterization and manufacture of proteins and polysaccharides. One of the first products produced using these techniques was cloned recombinant human insulin. Since its initial implementation, biotechnology has enabled the development of a large array of biological products that have therapeutic or vaccinal properties (Crommelin and Schellekens (eds), in: From clone to clinic, Kluwer Academic Publishers, Dorddrecht, The Netherlands (1990); The Biotol Team (eds), in: Biotechnology Innovations in Health Care. Butterworth-Heinemann Ltd, (1991); Reidenberg (ed), in: The clinical pharmacology of biotechnology products, Elsevier Science Publishers (1991)).
One of the biotechnology "work horses" are the bacterial host strains, which are used to house cloned genes and for the large scale production of the cloned genes or the products of said cloned genes. Examples of these bacterial host strains include HB101, DH5, DH5.alpha., DH5.alpha.MCR, DH10, DH10B, C600 or LE392 (Grant et al, Proc Natl Acad Sci (USA) 87:4645-4649 (1990); Sambrook et al (eds), in: Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1993)). These host strains are designed to stably harbor and express cloned genes.
A major problem, however, associated with using bacterial host strains is the process of removing bacterial LPS from the final products. The biological properties of LPS have been extensively investigated (Rietschel et al, FASEB, 8(2):217-225 (1994); Raetz, J Bacteriol 175(18):5745-5753 (1993); and Alving, Immunobiol, 187:430-446 (1993)). This molecule has powerful pyrogenic activity, so that in humans nanogram quantities of LPS can induce febrile responses, which are mediated by host proinflammatory cytokines IL-1, IL-6, and TNF-.alpha. (Mackowiak (ed), in: Fever: Basic Mechanism and Management. Raven Press, NY (1991); Abernathy and Spink, J Clin Invest, 37:219-226 (1958); Greisman et al, J Clin Invest, 43:1747-1757 (1964); Rietschel et al, supra; and Raetz, supra (1993)). For this reason, the United States Food and Drug Administration has strict guidelines on the level of LPS that is acceptable in biomedical products (Good Manufacturing Practices, in: U.S. Code of Federal Regulation 210-211; and Protection of Human Subjects, U.S. Code of Federal Regulation 50, Food and Drug Administration, CBER, Rockville Md.). Since all the currently available host strains produce pyrogenic LPS, this activity must be removed to acceptable levels, resulting in additional manufacturing costs.
2. Gene therapy and genetic immunization
The commercial applications of DNA delivery technology to animal cells are extremely broad and include delivery of vaccine antigens (Fynan et al, Proc. Natl. Acad. Sci., USA, 90:11478-11482 (1993); Katsumi et al, Hum Gene Ther, 5(11):1335-1339 (1994); Spooner et al, Int J Oncol, 6(6):1203-1208 (1995)), immunotherapeutic agents (Shillitoe et al, Eur J Cancer 30B(3):143-154 (1994); Hengge et al, Nature Genetics, 10(2):161-166 (1995); Vile and Hart, Ann Oncol, 5(Suppl 4):59-65 (1994); Miller et al, Ann Surg Oncol, 1(5):436-450 (1994); Foa, Baillieres Clin Haematol, 7(2):421-434 (1994)), and gene therapeutic agents (Darris et al, Cancer, 74(Suppl 3):1021-1025 (1994); Magrath, Ann. Oncol., 5(Suppl 1):67-70 (1994); Milligan et al, Ann. NY Acad. Sci., 716:228-241 (1994); Schreier, Pharma. Acta Helv., 68:145-159 (1994); Cech, Biochem. Soc. Trans., 21:229-234 (1993); Cech, Gene, 135:33-36 (1993); Dropulic and Jeang, Hum Gene Ther, U5(8):927-939 (1994); Sorscher et al, Hum Gene Ther, 5(10):1259-1277 (1994); Woo, Trends Genet, 10(4):111-112; Long et al, FASEB J., 7:25-30 (1993); Nabel et al, Hum Gene Ther 5(9):089-109 (1994); Manthorpe et al, Hum Gene Ther, 4(4):419-431 (1993); Mittal et al, Virus Res, 28:67-90 (1993); Setoguchi et al, Am J Respir Cell Mol Biol 10:369-377 (1994); and Rosi et al, Pharm. Therap., 50:245-254 (1991)).
In the aforementioned applications prolonged expression of the eukaryotic expression cassette once in the host tissue is highly desirable (Yang et al, J Virol, 69(4):2004-2015 (1995); Wicks et al, Hum Gene Ther, 6(3):317-323 (1995) and Alton et al, Nature Genet, 5(2):135-42 (1993)). Unfortunately, adenoviral vectors have proven to the highly immunogenic and induce a host response against cells containing these vectors (Yang et al, supra). This host response causes a more rapid clearance of the cells carrying adenovirus-delivered eukaryotic expression cassettes. Similarly, induction of inflammation at the site of "naked" DNA introduction or treatment with DNA encapsulated in cationic lipids can be deleterious to the elicitation of prolonged expression of the introduced eukaryotic expression cassette (Wicks et al, supra). A major cause of inflammation after introduction of DNA into the host is LPS, which co-purifies with DNA (Wicks et al, supra). LPS is a notorious biologically active molecule with potent pyrogenic properties (Mackowiak (ed), supra); Rietschel et al, supra; Raetz, surpa (1993); and Alving, Immunobiol, 187:430-446 (1993)).
Thus, sophisticated DNA purification procedures have been devised that remove LPS from the DNA prior to introduction into the host (Yang et al, supra; Nabel et al, supra; and Manthorpe et al, supra). These purification procedures involve removal of LPS with ionic detergents such as Triton X-114 or using polymyxin B columns (Yang et al, supra; Nabel et al, supra; and Manthorpe et al, supra). A weakness of this approach is that it adds additional cost to the commercial production of DNA and even after such purification procedures significant quantities of LPS remain associated with the DNA (Yang et al, supra; Nabel et al. supra; and Manthorpe et al, supra). This LPS will enter the host cells that receive the DNA encoding the eukaryotic expression cassette and exert its biological effects. Higher primates are more sensitive to LPS than laboratory rodents and under certain pathological conditions states of LPS hypersensitivity can be induced (Mackowiak (ed), supra); Abernathy and Spink, supra; and Greisman et al, surpa). Therefore, it is particularly important to produce DNA preparations that are free of LPS pyrogenic activity for applications in humans.
3. Bacterial strains in vaccine development
Inactivated and live attenuated bacteria are effective as vaccines (Holmgren et al, In: Vibrio cholerae and cholera. Wachsmuth et al. (eds), ASM Press Washington D.C., pp 415-424 (1994); Woodrow and Levine (eds), in: New Generation Vaccines, Marcel Dekker, New York (1990); and Cryz (ed) in: Vaccines and Immunotherapy, Pergamon Press New York (1991)) and as vector vaccines for the delivery of passenger antigens from other pathogens to the host immune system (Woodrow and Levine (eds), supra; and Cryz (ed) supra). In the role of vaccine vector, bacterial vaccines have the capacity to deliver bacterial antigens from bacterial pathogens, protozoan antigens, and viral antigens (Woodrow and Levine (eds), supra; and Cryz (ed) supra)). In order to be a successful vaccine or vaccine vector the inactivated or live attenuated bacterial vaccines or vector vaccine must be genetically stable, well-tolerated by the recipient host and stimulate humoral and able to T cell-mediated immunity in the recipient.
At present there is an attenuated strain of Salmonella typhi that is licensed for use as a live oral typhoid vaccine, strain Ty21a. Ty21a was prepared by successive exposures of wild type strain Ty2 to the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Consequent to this non-specific mutagenesis, Ty21a has multiple mutations including those in galE and causing Ty21a to Vi-negative. Ty21a is licensed in the USA where it is administered in a four dose immunization schedule.
Ty21a is an extremely well-tolerated live oral vaccine. Nevertheless, it suffers from many drawbacks that have been well-publicized in the scientific literature and it is well-recognized that a new and improved live oral typhoid vaccine is needed. The drawbacks of Ty21a include:
1) A Phase 1 volunteer study in which a defined S. typhi galE Vi-negative strain was ingested showed that the combination of a mutation in galE and lack of a Vi capsule are insufficient by themselves to fully attenuate wild type strain Ty2,the parent from which Ty21a was derived. Hence, the precise mutations that are responsible for the attenuation of Ty21a are not known and the task of defining the attenuating lesions in Ty21a might involve extensive genetic analysis as this strain was developed using non-specific chemical mutagenesis which introduces multiple point mutations in random locations on the target strain chromosome. PA1 2) Ty21a has been disappointing in attempts to utilize it as a live vaccine vector. Two Ty21a-based constructs have been evaluated in clinical trials, a candidate vaccine against Shigella sonnei consisting of Ty21a harboring a S. sonnei plasmid that results in expression of S. sonnei O antigen (Formal et al, Infect. Immun., 34:746-750 (1981)) and Ty21a having a plasmid allowing expression of the O antigen Vibrio cholerae O1 serotype Inaba (Forrest et al, J. Infect. Dis., 159:145-146 (1989)). In each instance results of clinical trials were disappointing (Levine and Tacket, in: Vibrio cholerae and cholera. Wachsmuth et al. (eds), ASM Press Washington D.C., pp 395-413 (1994)) and further clinical trials were abandoned. With each of these hybrid vaccines the limitations of Ty21a as a live vector were pointed out. PA1 Thus, there is clearly a need for an improved attenuated strain of S. typhi to serve as a live oral typhoid vaccine and as a live vector. PA1 induce type 2 T.sub.helper lymphocyte responses and mucosal secretory IgA responses. PA1 (i) auxotrophic mutations, such as aro Hoiseth et al, Nature, 291:238-239 (1981)), gua (McFarland et al, Microbiol. Path., 3:129-141 (1987)), nad Park et al, J. Bact., 170:3725-3730 1988), thy (Nnalue et al, Infect. Immun., 55:955-962 (1987)), and asd (Curtiss, supra) mutations; PA1 (ii) mutations that inactivate global regulatory functions, such as cya Curtiss et al, Infect. Immun., 55:3035-3043 (1987)), crp (Curtiss et al (1987), supra), phoP/phoQ (Groisman et al, Proc. Natl. Acad. Sci., USA, 86:7077-7081 (1989); and Miller et al, Proc. Natl. Acad. Sci., USA, 86:5054-5058 (1989)), phoP.sup.c (Miller et al, J. Bact., 17:2485-2490 (1990)) or ompR (Dorman et al, Infect. Immun., 57:2136-2140 (1989)) mutations; PA1 (iii) mutations that modify the stress response, such as recA (Buchmeier et al, Mol. Micro., 7:933-936 (1993)), htrA (Johnson et al, Mol. Micro., 5:401-407 (1991)), htpR (Neidhardt et al, Biochem. Biophys. Res. Com., 100:894-900 (1981)), hsp (Neidhardt et al, Ann. Rev. Genet., 18:295-329 (1984)) and groEL (Buchmeier et al, Sci., 248:730-732 (1990)) mutations; PA1 (iv) mutations in specific virulence factors, such as lsyA (Libby et al, Proc. Natl. Acad. Sci., USA, 91:489-493 (1994)), pag or prg (Miller et al (1990), supra; and Miller et al (1989), supra), iscA or virG (d'Hauteville et al, Mol. Micro., 6:833-841 (1992)), plcA (Mengaud et al, Mol. Microbiol., 5:367-72 (1991); Camilli et al, J. Exp. Med, 173:751-754 1991)), and act (Brundage et al, Proc. Natl. Acad. Sci., USA, 90:11890-11894 1993)) mutations; PA1 (v) mutations that affect DNA topology, such topA (Galan et al, Infect. Immun., 58:1879-1885 (1990)) mutation; PA1 (vi) mutations that alter the biogenesis of surface polysaccharides, such as rfb, galE (Hone et al, J. Infect. Dis., 156:164-167 (1987)) or via (Popoff et al, J. Gen. Microbiol., 138:297-304 1992)) mutations; PA1 (vii) mutations that modify suicide systems, such as sacB (Recorbet et al, App. Environ. Micro., 59:1361-1366 (1993); Quandt et al, Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al, App. Environ. Micro., 60:3746-3751 (1994)), hok, gef, kil, or phlA (Molin et al, Ann. Rev. Microbiol., 47:139-166 (1993)) mutations; PA1 (viii) mutations that introduce suicide systems, such as lysogens encoded by P22 (Rennell et al, Virol., 143:280-289 1985)), .lambda. murein transglycosylase Bienkowska-Szewczyk et al, Mol. Gen. Genet., 184:111-114 (1981)) or S-gene (Reader et al, Virol., 43:623-628 1971)); and PA1 (ix) mutations that disrupt or modify the correct cell cycle, such as minC (de Boer et al, Cell, 56:641-649 (1989)) mutation. PA1 (x) mutations that change the restriction-modification phenotype, such as deo, mcr, hsdR and hsdM (Grant et al, supra). PA1 (i) auxotrophic mutations, such as aro (Hoiseth et al, Nature, 291:238-239 (1981)), qua (McFarland et al, Microbiol. Path., 3:129-141 (1987)), nad (Park et al, J. Bact., 170:3725-3730 (1988), thy (Nnalue et al, Infect. Immun., 55:955-962 (1987)), and asd (Curtiss, supra) mutations; PA1 (ii) mutations that inactivate global regulatory functions, such as cya (Curtiss et al, Infect. Immun., 55:3035-3043 (1987)), crp (Curtiss et al (1987), supra), phoP/phoQ (Groisman et al, Proc. Natl. Acad. Sci., USA, 86:7077-7081 (1989); and Miller et al, Proc. Natl. Acad. Sci., USA, 86:5054-5058 (1989)), phoP.sup.c (Miller et al, J. Bact., 172:2485-2490 (1990)) or ompR (Dorman et al, Infect. Immun., 57:2136-2140 (1989)) mutations; PA1 (iii) mutations that modify the stress response, such as recA (Buchmeier et al, Mol. Micro., 7:933-936 (1993)), htrA (Johnson et al, Mol. Micro., 5:401-407 1991)), htpR (Neidhardt et al, Biochem. Biophys. Res. Com., 100:894-900 (1981)), hsp (Neidhardt et al, Ann. Rev. Genet., 18:295-329 (1984)) and groEL (Buchmeier et al, Sci., 248:730-732 (1990)) mutations; PA1 (iv) mutations in specific virulence factors, such as lsyA (Libby et al, Proc. Natl. Acad. Sci., USA, 91:489-493 (1994)), pag or prg (Miller et al (1990), supra; and Miller et al (1989), supra), iscA or virG (d'Hauteville et al, Mol. Micro., 6:833-841 (1992)), plcA (Mengaud et al, Mol. Microbiol., 5:367-72 (1991); Camilli et al, J. Exp. Med, 173:751-754 (1991)), and act (Brundage et al, Proc. Natl. Acad. Sci., USA, 90:11890-11894 (1993)) mutations; PA1 (v) mutations that affect DNA topology, such as topA (Galan et al, Infect. Immun., 58:1879-1885 (1990)) mutation; PA1 (vi) mutations that alter the biogenesis of surface polysaccharides, such as rfb, galE (Hone et al, J. Infect. Dis., 156:164-167 (1987)) or via (Popoff et al, J. Gen. Microbiol., 138:297-304 (1992)) mutations; PA1 (vii) mutations that modify suicide systems, such as sacB (Recorbet et al, App. Environ. Micro., 59:1361-1366 (1993); Quandt et al, Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al, App. Environ. Micro., 60:3746-3751 (1994)), hok, gef, kil, or phlA (Molin et al, Ann. Rev. Microbiol., 47:139-166 (1993)) mutations; PA1 (viii) mutations that introduce suicide systems, such as lysogens encoded by P22 (Rennell et al, Virol., 143:280-289 (1985)), .lambda. murein transglycosylase Bienkowska-Szewczyk et al, Mol. Gen. Genet., 184:111-114 (1981)) or S-gene Reader et al, Virol., 43:623-628 (1971)); and PA1 (ix) mutations that disrupt or modify the correct cell cycle, such as minC (de Boer et al, Cell, 56:641-649 (1989)) mutation.
Since the introduction of recombinant DNA techniques into the field of vaccine development it is now possible to attenuate strains with specific and precise genetic mutations that cause defined effects to the virulence of the target organism. Thus, to overcome this first shortfall of Ty21a, future strains must be made by precise genetic techniques.
Some mutations are known to attenuate wild type Salmonella thereby rendering said mutants promising (well-tolerated and protective) as live vaccines in mice and calves. These include strains harboring deletions in cya and crp (cya and crp constitute a global regulatory system in Salmonella) or in one or more genes (aroA, aroC or aroD) that encode critical enzymes in the aromatic amino acid biosynthesis pathway (Woodrow and Levine, supra).
Three such candidates have been assessed in Phase 1 clinical trials, including .chi.3927, a cya, crp mutant derived from wild type strain Ty2, CVD 908, a .DELTA.aroC, .DELTA.aroD mutant derived from Ty2 and CVD 906, a .DELTA.aroC, .DELTA.aroD mutant derived from wild type strain ISP 1820 (a minimally-passaged 1983 isolate from the blood culture of a Chilean child with uncomplicated typhoid fever) (Tacket et al, Infect. Immun 60:536-541 (1992); Tacket et al, Vaccine 10:443-446 (1992b); and Hone et al, J. Clin. Invest 90:412-420 (1992)).
These three live bacterial vaccine candidates were fed to adult volunteers in single doses of 5.times.10.sup.4 or 5.times.10.sup.5 cf, with buffer, in a randomized, double-blind clinical trial (Tacket et al, supra (1992); Tacket et al, supra (1992b); and Hone et al, supra(1992)). Significant febrile responses were observed in some recipients of strains CVD 906 or .chi..sup.3927, so further clinical trials with these strains were abandoned (Tacket et al, supra (1992) and Hone et al, supra(1992)). In contrast, CVD 908 did not cause notable systemic reaction, so that additional dose response studies were carried out with CVD 908 (Tacket et al, supra (1992); Tacket et al, supra (1992b)).
In subsequent Phase 1 clinical trials with CVD 908 in which adult volunteers were orally vaccinated with freshly-harvested vaccine organisms, the vaccine, was well-tolerated in single doses as high as 5.times.10.sup.7 and 5.times.10.sup.8 cfu (Levine, Session 57, American Society for Microbiology Annual Meeting, Washington D.C. (1995); and Levine, Keystone meeting on mucosal immunity, Keystone Colorado J Cell Biochem 19A:238 (1995)). However, when attention was turned to the preparation definitive formulations of CVD 908 to be made from fermentor-grown organisms, certain limitations of the aro mutants became readily apparent. The quandary faced in preparing a definitive formulation how to be able to grow the vaccine organisms in sufficient p-aminobenzoic acid (PABA) to obtain high yields but yet avoid retention of so much PABA in the lyophilate that the vaccine organism is capable of excessive growth in vivo (which might result in adverse reactions) (Levine, Session 57, American Society for Microbiology Annual Meeting, Washington D.C. (1995); and Levine, Keystone meeting on mucosal immunity, Keystone Colorado J Cell Biochem 19A:238 (1995). By carefully adjusting the concentrations of PABA and other aromatic metabolite; in the growth medium and by utilizing other specific quality control steps in the production process, it is possible to prepare vaccine lots with the desired properties (Levine, Session 57, American Society for Microbiology Annual Meeting, Washington D.C. (1995); and Levine, Keystone meeting on mucosal immunity, Keystone Colorado J Cell Biochem 19A:238 (1995).
Nevertheless, an ideal attenuated strain for use as a live vaccine would not be subject to such stringent production constraints. Rather, an ideal attenuated strain would be one that could be manufactured in large scale with simpler production methods and less stringent growth medium requirements than those necessary for aro mutants.
Thus, in light of this practicality issue with CVD 908 there is a need to develop new attenuated mutants of Salmonella that bear defined attenuating lesions, that are amenable to large-scale formulation and that retain the ability to stimulate high levels of T cell-mediated immunity as well as serum and mucosal antibody responses.
A similar phenomenon has been observed by investigators while developing live oral Vibrio cholerae strains. While live oral .DELTA.ctxA Vibrio cholerae strain CVD 103-HgR is well tolerated and immunogenic in volunteers (Levine and Tacket, supra), other .DELTA.ctxA mutants of Vibrio cholerae, which carry the identical mutation in the identical parent strain, have proven to be reactogenic (Levine and Tacket, supra). Thus, it is not clear what additional change causes CVD 103-HgR to be well-tolerated in volunteers. Further, Shigella flexneri 2a .DELTA.aro, .DELTA.virG mutant, CVD 1203 (Noriega et al, Infect. Immun 62: 5168-5172 (1994)) displayed impressive immunogenicity in volunteers but also caused mild diarrhea in a significant number of volunteers (Levine, Session 57, American Society for Microbiology Annual Meeting, Washington D.C. (1995); and Levine, Keystone meeting on mucosal immunity, Keystone Colorado J Cell Biochem 19A:238 (1995). This indicates that further attenuated derivatives of CVD 1203 must be developed before such mutants are acceptable for large scale clinical evaluation (Levine, Session 57, American Society for Microbiology Annual Meeting, Washington D.C. (1995); and Levine, Keystone meeting on mucosal immunity, Keystone Colorado J Cell Biochem 19A:238 (1995).
Bacterial host strains can also serve as vectors for the delivery of protective antigens cloned from other pathogens. As used herein the expression of "protective antigens" means antigens or epitopes thereof which give rise to protective immunity against infection by the pathogen from which they are derived.
The pathogens from which genes encoding protective antigens include protozoan (Sadoff et al, Science, 240:336-337 (1988)), viral (Wu et al, Proc. Natl. Acad. Sci. USA, 86:4726-4730 (1989)) and bacterial (Formal et al, supra; Elements et al, 46:564-569 (1984)) pathogens.
Additionally, Escherichia coli has been employed as a vaccine vector for the delivery of Shigella antigens in volunteers (Formal et al, Infect Immun 46:465-470 (1984)). However, these recombinant strains have proven to be reactogenic. More recently, Vibrio strains have been used successfully as vaccine vectors in animal models (Butterton et al, Infect Immun 63:2689-2696 (1995)). It is likely, therefore, that any well-tolerated and immunogenic bacterial vaccine will have the potential to serve as a vaccine vector. While the bulk of the documented data discusses the use of live oral vaccine vectors, inactivated bacterial vaccine vector are also feasible (Cardenas et al, Vaccine 12:833-840 (1994)).
A key step toward the development of a multivalent bacterial vaccine vector, will be the development of attenuated, non-reverting, and immunogenic bacterial vaccines strains.
4. Bacterial LPS and Lipid A
Under normal conditions, LPS is inserted in the outer surface of the outer membrane of gram negative bacteria Schnaitman and Klena, Microbiol Rev, 57:655-682 (199); and Makela and Stocker, In: Handbook of endotoxin volume 1, Elsevier Biomedical Press, Amsterdam, Rietschel (ed), pp59-137 (1984)). Complete or "smooth" LPS is composed of three main domains called lipid A, the O-antigen (also called the O-polysaccharide) and the core region, which creates an oligosaccharide link between lipid A and the O antigen (Schnaitman and Klena, supra; and Makela and Stocker, supra). The O-antigen is composed of oligosaccharide repeat units. The structure and number of these repeats varies depending on the bacterial species and growth conditions, typically ranging from one to fifty repeats (Schnaitman and Klena, supra; and Makela and Stocker, supra). Some bacterial genera, such as Neiseria spp., produce LPS that has low numbers of O-antigen repeats and therefore is referred to as lipooligosaccharide (LOS) simply to reflect this fact (Schnaitman and Klena, supra; and Makela and Stocker, supra).
The biological properties of LPS have been extensively investigated (Rietschel et al, supra and Raetz, supra (1993)). This molecule has powerful pyrogenic properties and in humans ng quantities of LPS can induce febrile responses (Mackowiak (ed), supra; Greisman et al, supra; Abernathy and Spink, supra; Rietschel et al, supra; and Raetz, supra (1993)). These febrile responses are mediated by host proinflamatory cytokines IL-1, IL-6, and TNF-.alpha., the secretion of which is induced by LPS (Mackowiak (ed), supra; Rietschel at al, supra and Raetz, supra).
The biologically active component of LPS is lipid A (Rietschel et al, supra; Verma et al, Infect Immun, 60(6):2438-2444 (1992); Alving, J Immunol Meth, 140:1-13 (1991); Alving and Richards, Immunol Lett, 25:275-280 (1990); and Richard at al, Infect Immun, 56:682-686 (1988)). Activity analysis of lipid A biosynthesis precursors or synthetic intermediates showed that various elements of lipid A are essential for pyrogenicity (Rietschel et al, supra; Raetz, supra). Lipid X and lipid IVa are completely non-pyrogenic precursor forms of lipid A (Wang et al, Infect Immun, 59(12):4655-4664 (1991); Ulmer et al, Infect Immun, 60(12):145-5152 (1992); Kovach et al, J Exp Med, 172:77-84 (1990); Rietschel et al, supra; and Raetz, supra).
Lipid X is a monosaccharide precursor of lipid A (Rietschel et al, supra; and Raetz, supra (1993)). Lipid IVa, a tetraacyl precursor of lipid A, is interesting in that it retains the ability to bind to host cell surfaces but has no pyrogenicity, suggesting that binding to host cell surfaces per se does not inpart this biological property (Wang et al, Infect Immun, 59(12):4655-4664 (1991); Ulmer et al, Infect Immun, 60(12):145-5152 (1992); Kovach et al, J Exp Med, 172:77-84 (1990) and Rietschel et al, supra).
5. The genetics of lipid A biosynthesis
The genetics of lipid A biosynthesis are well described (Raetz, supra; Raetz, Ann Rev Biochem 59:129-170 (1990); and Schnaitman and Klena, supra). The majority of mutations that prevent the biosythesis of lipid A, such as mutations in lpxA, lpxB, kdsA, kdsB, kdtA, are lethal as the biosynthesis of lipid A is essental for cell survival (Rick et al, J Biol Chem, 252:4904-4912 (1977); Rick and Osborn, J Biol Chem, 252:4895-4903 (1977); Raetz et al, J Biol Chem, 260:16080-16088 (1985); Raetz, supra (1990); Raetz, supra (1993); and Schnaitman and Klena, supra). For the most part, therefore, analysis of these genes has involved the use of temperature-sensitive mutants, which only display null phenotypes under non-permissive conditions (Rick et al, supra; Rick and Osborn, supra; Raetz et al, supra; Raetz, supra (1990); Raetz, supra (1993); and Schnaitman and Klena, supra). When grown under non-permissive conditions, lpxB, kdsA, kdsB, kdtA mutants accumulate non-pyrogenic precursor forms of LPS (to about 50% of the total LPS), such as lipid X (also called 2,3-diacyl-glucosamine-1-phosphate) or lipid IVa. Conditional-mutations in kdsA and kdsB prevent the biosynthesis of 3-deoxy-D-manno-octulsonic acid (KDO) and conditional-mutations in kdtA prevent the transfer of KDO to lipid Iva (Rick et al, supra; Rick and Osborn, supra; Raetz et al, supra; Raetz, supra (1990); Raetz, supra (1993); and Schnaitman and Klena, supra). The absence of KDO moieties on lipid IVa prevents further acylation of lipid IVa resulting in the accumulation of this molecule when KDO synthesis is blocked. The necessity to add KDO to lipid IVa prior to completion of lipid A biosynthesis is further demonstrated by the fact that drugs designed to block KDO synthesis are highly toxic to gram negative bacteria Rick et al, supra; Rick and Osborn, supra; Raetz et al, supra; Raetz, supra (1990); Raetz, supra (1993); arid Schnaitman and Klena, supra). Conditional-mutations in the lpxA gene result in a 10-fold reduction of lipid A biosynthesis under non-permissive conditions by preventing transfer of .beta.-hydroxymyristate to UDP-GlcNAc, thereby preventing the synthesis of uridyldiphosphate-2,3-diacyl-glucosamine. Mutations in lpxA cause rapid cessation of growth and therefore the LpxA protein is a potential target for drug therapy. Further conditional-lethal mutants in lipid A biosynthesis also include lpxC and lpxD (Raetz, supra (1993)), which are necessary for the biosynthesis of uridyldiphosphate-2,3-diacyl-glucosamine. Recent evidence showing that ssc mutants (analogous to lpxD) of Salmonella typhimurium accumulate a pentaacyl form of lipid A indicates that this gene is also involved in lipid A biosynthesis.
There is indirect evidence that mutations in htrB and msbB may influence the biosynthesis of lipid A (Karow et al, J Bacteriol 173:741-750 (1991); Karow and Georgopoulos, J Bacteriol 174:702-710 (1992)). These mutants are temperature sensitive and LPS isolated from these mutants stains less intensely on silver-stain gels (Karow and Geogopoulos, supra). The basis for the temperature-sensitive growth phenotype of the htrB and msb mutants has remained cryptic (Karow and Geogopoulcas, supra). There has been speculation that these mutants produce defective lipid A precursors (Karow and Geogopoulos, supra). This was based on the observation that ammonium cationic compounds enabled these mutants to grow in non-permissive temperatures (Karow and Geogopoulos, supra). These investigators proposed that the ammonium cationic compounds influenced the intermolecular interaction between LPS molecules in the outer membrane. This observation is supported by recent data showing that an htrB mutant of Hemophillus influenzae produces modified LOS structures (Lee et al, Infect Immun 63:818-824 (1995); Lee at al, In: Abstracts of the American Socienty for Microbiology, ASM Washington D.C., p206(B-234) (1995)).
However, none of these investigators provided any direct evidence that htrB and msb mutants could produce substantially pure non-pyrogenic LPS. More importantly, these investigators did not show that these mutants would have the surprisingly broad biotechnology application described herein.
6. Summary of background
There is a need for non-pyrogenic bacterial host strains that can be used to produce non-pyrogenic DNA, proteins, polysaccharides, vaccines, and vaccine vectors. The present invention describes a novel and unexpected finding that gram negative bacterial mutants can be continuously grown in the presence of quaternary cationic compounds under non-permissive growth conditions and accumulate substantially pure non-pyrogenic lipid A precursors.